Brain metastasis (Brain-M) is considered the cause of death in half of metastatic melanoma patients and remains mostly incurable. Moreover, in this new era of more effective melanoma treatment, patients with systemic disease frequently survive long enough for melanoma cells to trespass the blood-brain barrier to establish Brain-M. Our central hypothesis is that radiation of melanoma Brain-M leads to up-regulation of immune checkpoint ligands and makes the Brain-M more responsive to immune checkpoint inhibitors. However, determining the best combination of therapies for patients with Brain-M is severely limited by: 1) A lack of clinically relevant humanized Brain-M preclinical models to test rational combinations, a prerequisite for moving into clinical trials, and 2) A limited understanding of the effect of radiation on tumor immunity and/or immune checkpoint ligands on Brain-M human tissues. To test our hypothesis, we will establish a novel preclinical humanized melanoma Brain-M model. We will also use our unique melanoma biospecimen database to test the effects of brain radiation on melanoma Brain-M. Our preliminary data, using our unique human specimen resources, demonstrate that radiation of melanoma Brain-M significantly up-regulate PD-L1 compared to non-radiated Brain-M. However, our proof of concept pilot experiment needs to be verified and validated using a larger sample size. We will test our hypothesis by pursuing the following aims: Aim 1: To develop a humanized preclinical model for melanoma Brain-M and exploit the potential of the combination of immune checkpoint inhibitor anti-PD1 with radiation. We propose to develop a humanized model of melanoma Brain-M that correctly recapitulates and addresses the specificity of the human immune system. We will then exploit anti-PD1 as an immunological adjuvant to radiation therapy in the humanized Brain-M mouse model, testing the hypothesis that whole-brain radiotherapy (2GyX5) to the melanoma Brain-M makes the tumor more responsive to immune check point inhibitors (e.g., anti-PD1) by up- regulating immune checkpoint ligands (e.g., anti-PD-L1). We will quantify tumor infiltrating lymphocytes (TILs), measure the expression of immune checkpoints and their ligands, and use cytokine beads array and ELISA to measure the impact of radiotherapy on cytokines produced. Aim 2: To characterize radiation-induced immune modulation of melanoma Brain-M in human specimens. We will test the hypothesis that radiation to the brain up-regulates immune checkpoint ligands in human Brain-M tumor tissues as well as changes the profile of secreted cytokines. We will compare 2 groups of Brain-M tissues in our NYU Melanoma biospecimen bank: 1) resected post-radiation and 2) resected with no previous exposure to brain radiation. Pilot analysis of these human samples supports that irradiated Brain-M up-regulate PD-L1. Further immunologic parameters will be measured in these Brain-M specimens as well as in sera from the same patients, as well as correlated with clinical response to radiation therapy and outcome.